LEE- Çevre Biyoteknolojisi- Doktora

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Lipid production by Yarrowia lipolytica growing on food waste

(Graduate School, 2023-05-17) Khaligh Salimi, Soodeh ; Altınbaş, Mahmut ; 501152806 ; Environmental Biotechnology

Biodiesel production from plants and vegetable oils or different organic wastes as feedstock for microorganisms, can helps to decrease the consumption of fossil fuels and generation of greenhouse gases along with improve the economy. Applying organic wastes as feedstock for oleaginous yeasts is an economic technique in order to replace fossil-derived diesel with biodiesel as a clean and green fuel. Using food waste (FW) as a rich organic carbon source for cultivation of oleaginous yeast is considered as a promising environmentally friendly approach to achieve microbial lipid as the source of biodiesel. In this thesis, FW was collected from refectory of Istanbul Technical University. It contained cooked and uncooked food which dried and filtered after collection. Dark fermentation process was carried out with the collected FW and rumen microorganisms as inoculum. The rumen was taken from sheep stomach. The output of fermentation process was collected and applied as substrate for cultivation of oleaginous yeast Yarrowia lipolytica. The Soluble COD (SCOD) and TKN concentrations of fermented food waste (FFW) were 48.400 ± 0.49 and 0.907 ± 0.01 g/L, respectively and pH of the medium was 5.44 ± 0.05. Different concentrations of FFW (diluted with distilled water) were applied as growth medium of Y. lipolytica. The medium which was applied with no dilution was identified as the optimum one. In all applied mediums the growth was monitored as optical density (OD600) and the highest OD of 36.11 was observed in medium with FFW with no dilution. Nitrogen is considered as an essential nutrient for synthesis of cell materials and metabolites by microorganism. In terms of nitrogen depletion along with excess amount of carbon in the culture, carbon uptake rate is limited that causes metabolic activities shift towards lipid storage instead of cell proliferation. This approach was used in this thesis to increase lipid content of Y. lipolytica cultivated on FFW. Different carbon sources of glucose, glycerol, and potassium acetate along with five different COD/TKN ratios of 75, 100, 125, 150 and 175 were selected. Carbon sources were added to the medium in early stationary phase to increase carbon concentration of FFW and obtain favorable COD/TKN ratios. In order to identify the appropriate fermentation time to collect the biomass to evaluate its lipid content, biomass samples were collected at both early and late stationary phase in YPD (the optimum growth medium for yeast growth) and FFW medium. The results indicated lipid content of 21.54 ± 1.4 and 14.97 ± 0.51% lipid along with biomass concentration of 9.63 ± 0.24 and 8.34 ± 0.82 g/L in early and late stationary phase respectively, for YPD medium. These values were 19.5 ± 0.5 and 15.52 ± 0.31% lipid content along with 8.31 ± 0.51 and 7.10 ± 0.34 g/L biomass generation for Y. lipolytica cultivated on FFW medium. Results illustrated the early stationary phase as the optimum fermentation point to obtain highest lipid content and microbial cell because the biodegradation of yeast cell and intracellular lipid is occurred by time during stationary phase. This biodegradation caused drop of intracellular lipid content and yeast cell quantity at the end of stationary phase. In COD/TKN 75, lipid content of biomass was 26.7 ± 0.5, 34.2 ± 1.12 and 33.2 ± 1.59% with biomass concentration of 9.50 ± 0.37, 8.40 ± 0.72 and 9.32 ± 0.12 g/L for mediums contain glucose, glycerol, and potassium acetate, respectively. By increasing the ratio to 100, lipid content of medium with glucose increased to 29.2 ± 0.28, in medium contains glycerol lipid content was 34.3 ± 2.16 and in medium with potassium acetate the lipid content was slightly decreased to 31.2 ± 1.60%. In COD/TKN ratio of 125, the significant lipid content of 42.2 ± 1.72% in the medium supplemented with glycerol was measured. The other mediums contain glucose and potassium acetate have intracellular lipid content of 38.7 ± 0.35 and 34.7 ± 3.1%, respectively. Biomass concentration was measured as 18.52 ± 1.97, 12.95 ± 1.95 and 17.60 ± 0.75 g/L in culture sets supplemented with glucose, glycerol, and potassium acetate, respectively. In COD/TKN ratios 150 and 175, the amount of accumulated lipid and generated biomass were decreased that demonstrated the adverse effect of high concentration of carbon source on intracellular lipid accumulation of the cell. In these two ratios the amount of lipid content and cell concentration was 36.7 ± 1.3 and 9.77 ± 0.97; and 38.1 ± 3.0% and 11.57 ± 0.77 g/L, respectively. Highest lipid concentration of 7.61 ± 0.17 g/L was observed in medium with potassium acetate in COD/TKN 100 that is related to high biomass concentration in this experimental set. Lipid concentration started to decrease in higher COD/TKN ratios and dropped to 3.59 ± 0.13 g/L in COD/TKN ratio 150 with glycerol. Although the final pH value of the mediums with COD/TKN 125 and lower was always over 8, in ratios over 125 the pH was dropped to 4.3. This pH indicated formation of secondary metabolites such as organic acids in higher ratios that was due to high glycerol concentration in the mediums. This metabolic shift from lipid accumulation to organic acid generation led to the pH drop of the batch cultures. In next step of the thesis, Yeast Extract (YE), Iron Sulphate (IS) and Trace elements Solution (TS) was supplied to FFW along with glycerol as second carbon source to boost the lipid content further. Firstly, various amount of mentioned components were added to FFW to investigate the optimum concentration of each one to enhance lipid content of the cell. The concentrations of 1000 mg/L YE, 150 mg/L IS, and 5 ml/L TS were identified as the optimum. Different dual and triple combinations of these components along with glycerol were added to the mediums. In COD/TKN 150, accumulated lipid was reached to 44.72 ± 0.31% in the medium contains YE+IS+TS. Accumulated lipid reached to its maximum in ratio 175 i.e., 45.94 ± 0.21% in experimental set supplemented with YE+IS+TS. This value was the highest lipid content obtained in this thesis and by increasing the COD/TKN ratio over 175, the lipid accumulation dropped significantly. The highest concentration of lipid was 7.12 ± 0.12 g/L obtained in ratio 175 in medium supplied with YE+TS. The highest cell production was measured in the same culture with biomass concentration of 16.67 ± 0.27 g/L. Investigation of metabolic behavior of Y. lipolytica in this thesis revealed that in general, by increasing concentration of organic carbon in the medium, the carbon consumption is enhanced as well. In ratio 75, the highest COD consumption was measured in culture contains glycerol as 30.11 g/L COD. In ratio 100, the COD consumption of medium contain glycerol was increased slightly to 32.54 mg/L COD. In ratio 125, the COD consumption of 38.84 g/L was observed that caused the lipid storage of 42.2 ± 1.72% w/w in the cell. By increasing the COD/TKN ratios to 150 and 175, the microbial lipid content decreased although COD consumption was increased. This demonstrated the metabolic shift of Y. lipolytica from lipid generation to secondary metabolite production which caused drop of lipid biosynthesis. By adding YE, IS and TS to the medium, in ratio 150 the lipid content enhanced to 44.72 ± 0.31% with COD consumption of 48.37 g/L and in ratio 175, highest lipid content of 45.94 ± 0.21% was obtained by COD consumption of 49.63 g/L. However, decreasing pH value in experimental sets with COD/TKN over 125 indicated the generation of secondary metabolites in the medium. Additionally, in ratio 200 and 225 despite increase in COD consumption in the culture, the amount of stored lipid was reduced. Gas chromatography analyses of accumulated lipid revealed the fatty acid (FA) composition of stearic acid (C18:0), linoleic acid (C18:2), pentadecanoic acid (C15:1), palmitic acid (C16:0) and heptadecenoic acid (C17:1), similar to the FA profile of plant oil and appropriate for biodiesel generation. This thesis presented the efficient conversion of FFW and glycerol to considerable amount of microbial lipid. It indicated that FW and glycerol as two available and economic carbon sources can be evaluated as feedstock for intracellular lipid accumulation of Y. lipolytica. Additionally, this study demonstrated that the two-stage batch cultivation method by using FFW as initial carbon source and glycerol as the second one, can improve the lipid content of the microbial cell in significant amount. This thesis provides not only an economic waste treatment strategy, but also a sustainable and profitable method of microbial oil production by using carbon rich wastes as substrate for oleaginous yeast.
Environmental stresses applied to microalgae for high lipid production

(Graduate School, 2023-07-17) Polat, Ece ; Altınbaş, Mahmut ; 501122801 ; Environmental Biotechnology

Natural resources are a major part of global economy, and a number of regulations have been implemented to increase the demand for renewable energy resources. There is a great deal of potential in biomass energy, since it is a low-cost and sustainable form of energy. In recent years, there has been significant research on the use of vegetable oil in the production of biodiesel as a competitive fuel to oil-based diesel, which results in significantly less environmental damage. The use of renewable energy sources such as biomass, water, and solar is becoming increasingly prevalent in an attempt to minimize environmental problems and emissions from fossil fuels. A very attractive solution would be to use microalgae as a fuel source since they can store high levels of lipids and can be fueled by solar energy. Due to their low space requirements and high lipid content, microalgae are preferred by researchers over plants and other types of energy sources. The majority of current studies on microalgae focus on macroalgae that produce high levels of lipids. Accordingly, stress conditions, adaptations, or genetic manipulations are being investigated. Autotrophs and heterotrophs produce a large amount of lipids, making them ideal candidates for biofuel production. In spite of this, fossil fuel production still has a relatively low cost in comparison to the production of fuel from algae. Due to these reasons, using microalgae as biodiesel is not economically feasible. Furthermore, in order for microalgae to be economically competitive, it is necessary to increase lipid production efficiency. It is likely that the increased efficiency of lipid production has been attributed to both an increase in the amount of lipid produced and a concurrently high rate of biomass production. According to previous studies, microalgae under stress induce biomass production, biomass inhibition, or significant changes in the structure of biochemical substances. They produce higher levels of lipids, proteins, carbohydrates, and fatty acids, as well as higher quality methyl ester fatty acids. It has been determined that microalgae that are intended for use as biofuels are required to produce a high level of biomass, a high level of lipid, and a high level of fatty acid methyl ester quality. A major objective of this study is to reduce the cost of producing lipids that can be used in the production of biodiesel, which can be used in place of fossil fuels. Thus, an extremely significant step will be taken to obtain microalgae efficient enough to compete economically with fossil fuels. In this study, the microalgae, mainly Auxenochlorella protothecoides, which have both heterotrophic and phototrophic growth properties, were investigated to obtain microalgae with suitable biomass, lipid, and lipid composition. The thesis is organized into ten chapters. In order to evaluate the sustainability of microalgae biodiesel production under stressful environmental conditions, various nutrient stress factors will be examined in relation to growth parameters, such as growth kinetics, biomass, lipid and fatty acid methyl ester composition, chlorophyll content, and carotene content. Based on the results obtained, it was determined whether or not biodiesel-quality lipids could be obtained. In the next step, multiple effects of different stress factors were examined, and optimum parameter values were derived using the surface response approach. The final section of the study examined the effect of the two-stage growth process on microalgae lipid production. Studying the addition or deprivation of ferrous sulfate at different concentrations revealed that only a concentration of 0.2 mM and 14.4 mM ferrous sulfate maintained the lipid in high-quality biodiesel. Additionally, Auxenochlorella protothecoides displayed growth properties even at a concentration of 21.6 mM iron sulfate. At 1.08 mM ferrous sulfate concentration, the highest biomass was obtained (1520 mg / L), while the highest saturated fatty acids were obtained at 1.44 mM ferrous sulfate concentration. Despite no significant variation in lipid production, iron deprivation led to the greatest amount of lipid (59.6%). When nitrogen starvation, deprivation, and excess addition were applied, only biomass grown under 0.8 mM NH4Cl met the biodiesel standard. In this case, the amount of lipid measured is 53.8%. However, the biomass obtained in this case is 1.7 times lower than that obtained in a nitrogen-containing medium. This study illustrates that biomass decreases under stress in response to an increase in lipid content. Through participation in various cell metabolic pathways via changing some enzyme activities, plant hormones are able to change the metabolism of plants, including acceleration or deceleration of cell growth and stimulation of cell biochemical products. Abscisic acid hormone is an inhibitory growth hormone used in plants. Adding this hormone to the growth environment of Auxenochlorella protothecoides may lead to more lipid production. According to this theory, abscisic acid was added in a different medium than in other studies, which served as a source of carbon for glycerol. Under 2.5 µM to 40 µM abscisic acid concentrations, microalgal lipids met high-quality biodiesel standards. It was also concluded that a concentration of 2.5 µM abscisic acid promotes growth. Additionally, high levels of lipids were found at concentrations of 2.5 µM and 10 µM abscisic acid. This thesis also examines the effects of multiple stress factors on microalgae lipids and biomass by using the surface response method and experimental results were correlated with quadratic equations, and optimum conditions for maximum biomass and maximum lipid were determined. For this purpose, in the first study, the acetate parameters known as the carbon source and pH as an indicator of hydrogen ion concentration were selected as variables. The growth experiments of batch microalgal growth have been conducted in different pH and acetate-containing media, which were modeled in three dimensions using surface response methodology, resulting in situations where high biomass and high lipid conditions were achieved and obtained as a result of this model. A second study concerning multiple stresses examined the effects of magnesium deprivation and sodium chloride salt on chlorophyll, carotene, biomass, lipid, protein, and carbohydrate parameters in growth media. Biomarkers of stress such as reactive oxygen species and malondialdehyde, a product of lipid peroxidation, confirmed these changes. Surface response methodologies were used to obtain three-dimensional graphs of the results, and magnesium and sodium chloride concentrations, which were likely to maximize biomass and lipid production, were calculated. An increase in lipid content while a decrease in biomass is generally insufficient to develop a strain with improved biodiesel properties. A robust strain with high lipid and biomass content may be obtained through chemical mutagenesis such as ethyl methane sulfonate. In this study, suitable mutants have been selected considering a selective environment which is a kind of ACCase inhibitor herbicide. In the final stages of this thesis, this study examined the effect of different forms of nitrogen (nitrate and ammonia) on the growth of mixed microalgal cultures in anaerobic digestate. The Biodiesel properties of lipids obtained from microalgae cultured under salt and iron stress were evaluated for their suitability as biodiesel feedstock. This thesis revealed that it is necessary to develop systems that make biomass available as a source of energy so as to reduce the use of fossil fuels as an alternative energy source. Furthermore, results were obtained to support microalgae-based biodiesel production, which would contribute to the lack of literature on multiple stress and singular stress.
Improving raceway reactor productivity via vortex induced vibrations for cost effective microalgae production

(Graduate School, 2023-09-15) Akca, Mehmet Sadık ; İnanç, Bülent ; 501152801 ; Environmental Biotechnology

Microalgae research has been becoming more and more common in the last decades due to a number reasons including need for sustainable energy and fuel production, concerns about climate change and orientation of people to biobased products. Microalgae is considered as an excellent feedstock to meet peoples future demands in these fields. Microalgae converts inorganic carbon into sugar using radiative energy in the process so called "photosynthesis". It can grow on non arable land and does not compete with agricultural food products, can assimilate waste products such as flue gas and wastewater and convert them into biomass. Microalgae cultivation is carried out for both remediation of waste streams and commercial purposes. Algal wastewater treatment is a hot topic in environmental engineering and poses several advantages compared to conventional wastewater treatment processes such as reduction of aeration costs. While algal nutrient removal is more common, certain microalgae species can assimilate organic carbon, making it an interesting alternative to activated sludge process. However, algal wastewater treatment is limited to several community scale facilities. Commercial scale algal biomass production is dominated by food and feed industry. While Spirulina and Chlorella are the most commonly cultivated species, cultivation of Haematococcus and Dunaliella is common due to their ability to synthesize high value products such as astaxhanthin and carotenoids. Microalgae based biodiesel is considered as among the main candidates to replace fossil fuels as algae can accumulate lipids up to 70% their dry weight and it can be said that most of the research effort involving microalgae is towards this subject. However, algal biodiesel is not economically feasible yet due to high costs of cultivation, harvesting and other downstream processes. Microalgae cultivation systems are generally classified as open and closed systems. Closed systems offer a more controlled environment with higher light availability; light paths being couple of centimeters to 10 cm. Microalgae growth rate and biomass concentration is higher in this type of systems; however much higher capital and operating costs, as well as upscaling issues strongly limits their utilization. Open systems on the other hand are much easier to build and operate. Raceway ponds is the most common microalgae cultivation system. A raceway can be defined as an oblong channel where culture medium is most commonly circulated with the help of a paddlewheel. A single pond can occupy an area up to 4 hectars. Depth of the pond is kept 20-30 cm to ensure light penetration and flow velocity is typically 0.2-0.3 m/s. While 90% of commercial scale microalgae production is carried out in raceway ponds it has strong disadvantages compared to closed photobioreactors such as limited light availability and vulnerability to environmental and climatic conditions. Among these, light availability is perhaps the most important bottleneck for optimization of low cost microalgae biomass production. Limited light availability results from very limited vertical mixing in long straight channels of raceway ponds. Improving vertical mixing can be achieved by introducing more turbulent to flow by increasing flow velocity, which is energy intensive. Thus, energy efficient systems for improving vertical mixing and creating light dark cycles in raceway ponds is a strong necessity for making algal products more economically attractive. xxii Aim of this thesis to improve vertical mixing in raceway ponds without increasing operational costs. Method to improve vertical mixing is implementation of vortex induced vibrations. Vortex induced vibration is a form of flow induced motion whereby a body becomes excited, with vortices shed from its surface. These vortices, when they shed and leave the surface, exert force on the cylinder. When a vortex separates from the top part, the cylinder feels a downward force. When it separates from the bottom, the direction of the force is then upwards. When the cylinder is allowed to move in the direction perpendicular to the flow, cylinder moves up and down. This is a periodic motion and will last forever as the fluid continues to flow. Vortex induced vibrations make use of the flow energy and convert this power of the fluid to oscillate the cylinder. Within the scope of the thesis vortex induced vibration systems are used to improve vertical mixing in raceway ponds without any additional energy input. Vortex induced cylinder oscillation requires flow uniformity along the width of the channel where the system was implemented. For this, first, flow field of existing raceway pond at the roof of ITU Environmental Engineering Department was numerically investigated using CFD code, to see if it is available for implementation of vortex induced vibration systems. Flow velocity is kept as 0.3 m/s. Paddlewheel was removed from the domain to decrease computational effort and k- ɛ was chosen as turbulence model. In the CFD analyses, the raceway pond was modified with one, two and three flow deflectors and width of the central divider was increased to 5 and 10 cm. It has been seen that by installing 3 semi-circular flow deflectors in the bends of the pond, uniform flow along channel width could be achieved. Existing raceway pond was modified in this way and vortex induced vibration system, which consists of a cylinder with 6 cm diameter and two springs was installed to pond. Continuous cylinder oscillation was achieved with 6.5 cm vertical amplitude and 1.24 s-1 oscillation frequency while water level was 0.3 m. Impact of this cylinder motion on vertical mixing was numerically analyzed using CFD code. To simulate the cylinder oscillation, governing equations of vortex induced vibration was implemented to model as user defined function. Flow velocity was kept as 0.3 m/s as in the experiments and k- Ω SST was chosen as turbulence model. Model was run under steady conditions until the dynamic equilibrium was reached. After this, model was run for 10 seconds to investigate VIV motion. Model output revealed that vertical motion of flow covered 2/3 of pond depth. Cylinder oscillation directs flow upwards with a magnitude of 0.3 m/s and creates high frequency light dark cycles to effectively utilize so called flashing light effect. Light to dark cut off point was assumed as 3 cm below culture surface and average frequency of L/D cycles in the first 60 cm downstream of the cylinder for uppermost, neutral and lowermost cylinder positions were calculated as 21.17 s-1, 5.28 s-1 and 2.33 s-1, respectively. Pure culture of Chlorella vulgaris was grown comparatively to assess the effect of VIV on biomass production capacity. Culture was first grown under laboratory conditions in 10 L plastic bottles. Temperature was kept constant at 28 oC and Bald's Basal Medium was used as growth medium. Culture was grown for 1 week in laboratory and after that transferred to open ponds at the roof of ITU Environmental Engineering department. Culture was further grown for 1 week to acclimate outdoor conditions and diurnal cycle. VIV system was removed from the pond and culture was grown in two identical ponds for additional one week two make sure identical ponds demonstrated the same performance in terms of biomass production capacity. VIV system was implemented to one of the ponds at the end of this week and comparative cultivation xxiii with and without VIV system was carried out for one week. Biomass growth was monitored by optical density measurement under wavelength of 540, 690 and 750 nm. Experiments revealed that VIV increased biomass production capacity in the pilot scale raceway pond with 3 m channel length and 1 m total width by over 20%. Amplitude response of cylinder achieved in the pilot scale raceway pond for 0.3 m/s flow velocity was lower compared to literature. To investigate the reason of this and to investigate effect of VIV on vertical mixing and light dark cycles in the raceway pond in detail, flow visualization technique was applied. Particle image velocimetry using LED illumination was applied under experimental conditions mentioned above. Frame rate of PIV camera was 165 FPS and focal length was 35 mm. Flow visualization experiments without the VIV system revealed that flow velocity decreases through pond depth in the paddlewheel driven system with a 4.5 cm bottom clearance. Distribution of horizontal flow velocity could be modeled with 2nd order polynomial. This uneven distribution of flow velocity through the depth suppresses cylinder motion which resulted in lower amplitude response compared to literature. Several other equipment such as archimedes pumps, centrifugal pumps, airlift pumps and propellers are proposed to replace the paddlewheel for further exploitation of effect of VIV motion on vertical mixing and thus light availability and biomass production capacity. Indeed, it has been reported that propellers and airlift pumps are more energy efficient than paddlewheels. Furthermore, paddle induced circulation would become more disadvantageous when culture depth was increased due to mechanical reasons. Flow field in the raceway pond when vortex induced vibration system was implemented was analyzed using particle imaginary technique. Vertical component of flow was in accordance with CFD analyzes in general. 75 cells were selected at the downstream of VIV cylinder and were tracked for 20 cm in horizontal direction, until they disappeared from the other side of cameras projection area. Initial cell positions were set as three equidistant planes through the depth of the raceway channel to represent the average situation. Flow visualization experiments revealed that 33% of selected cells entered high frequency light dark cycles with the help of VIV. Average frequency of light dark cycle was found to be 35.69 s-1 with a light fraction of 0.49. 44% of cells entered light limited zone from dark zone as a result of VIV motion. Pilot scale RWP has 3 m channel length, which means, compared to full scale facilities, cells pass through paddlewheel, where vertical mixing happens, more frequently. In other words, pilot scale RWP is more effectively mixed compared to full scale systems. By installing one VIV cylinder, it can be said that a 2nd vertical mixing point was created in the pond. On the other hand, real scale RWPs have much higher channel lengths, thus effect of paddle induced vertical mixing in these systems would be less pronounced. In these long channel sections, cells near the surface will become "over- charged" after a certain period of time. On the other hand, cells at lower parts of pond depth will reside in photobiologically inactive parts of the pond for a prolonged period. As indicated above, VIVs can cycle cells between photobiologically active and inactive parts of channel and increase number of cells that perform photosynthesis in one circulation around pond. Thus, it is believed that the effect of VIV could be more pronounced in larger ponds.
Valorization of lignocellulosic waste by oleaginous yeast

(Graduate School, 2023-09-15) Ünver, Hülya ; Altınbaş, Mahmut ; 501132806 ; Environmental Biotechnology

In this study, biomass and lipid production capacity of oleaginous yeast Yarrowia lipolytica grown on wood hydrolysate was invesitgated. The demand in biofuel market prompted the third generation of bio-oil production via oleaginous species. Being noncompetitive with food resources unlike other second generation oil plants, microbial oil production provides a chance for the valorization of a variety of nutrient sources abundant in the environment such as waste and waste streams, industrial, agricultural, and forest residues. Among these resources, the potential of woody biomass as a feedstock for biotechnological species has recently increased efforts for its utilization to produce valuable microbial products due to the high content of lignocellulosic (LC) sugars for their bioconversion.
Investigation of treatment performances and energy recoveries from a real textile wastewater in conventional and high-rate MBR processes

(Graduate School, 2024-01-11) Yılmaz, Tülay ; Çokgör, Emine ; Şahinkaya, Erkan ; 501192805 ; Environmental Biotechnology

Textile industry wastewater, which is among the most water-consuming sectors worldwide, is extremely hazardous for receiving water bodies due to its toxic and complex structure. Many studies have been conducted involving physical, chemical, biological and combined processes for the textile wastewater treatment, but none of them alone is sufficient to meet discharge standards, and each process requires higher investment and operating costs. Additionally, energy-neutral plants should be expanded to ensure circular economy during the textile wastewater treatment, and the treated wastewater should be appropriate to be used for further reuse processes. Aerobic membrane bioreactors (MBRs) have been widely preferred in the textile wastewater treatment due to their ease of operation, lower volume requirement, and providing higher quality effluent compared to traditional biological methods. However, aerobic MBRs require higher energy input to both supply the oxygen requirement of microorganisms and minimize membrane fouling. In the treatment of textile industry wastewater, a single aerobic process is not sufficient to remove organic matters, azo and reactive dyes (anaerobic/aerobic combined system are needed) and nitrogen-based pollutants (nitrification and denitrification processes are needed). In addition, processes selected should be less-energy consuming due to high volume of textile industry wastewater. Considering that water resources are decreasing globally today, it is not enough to treat textile industry wastewater and their recycling is also extremely important. Therefore, sustainable and practical treatment strategies should be developed for textile industry. This thesis aims to investigate the treatment potential of two different biological processes with different advantages for real textile wastewater. First of all, the treatment performance of the real textile wastewater and the recovery of organic matter that can be preferred as a raw material source for biomethane generation were investigated in an aerobic high-rate MBR including a hollow fiber (HF) ultrafiltration membrane (UF) with a pore size of 0.04 µm. In the high-rate MBR process, it was aimed to recover organic matters rather than oxidation at short (0.5-5 days) sludge retention times (SRTs) and hydraulic retention times (HRTs). Additionally, the effects of SRT/HRT ratios on membrane filtration performance, sludge characterization, sludge production, and the energy requirements for aeration were examined, and all parameters were compared with a conventional long SRT aerobic MBR system operated in parallel. In another MBR process equipped with an HF-UF membrane, the feasibility of intermittent aeration strategy for simultaneous nitrification and denitrification to remove nitrogen-based pollutants and for energy minimization were investigated. Additionally, the effects of different aeration patterns on membrane filtration performance, sludge characterization, sludge production and energy requirements for aeration, and specific removal rates were studied. Within the scope of this thesis, all studies were carried out in four stages. In the first stage, textile wastewater treatment performances of conventional MBRs were investigated at SRTs of 30, 20 and 10 d. During this stage, two identical MBR systems were run, one at SRT for 30 days (MBR-1), and the other at SRT for 20 and 10 days (MBR-2), respectively. The average total chemical oxygen demand (COD), color and total dissolved nitrogen concentrations of textile wastewater used in the study averaged 927±277 mg/L, 910±287 Pt–Co and 39±10 mg/L, respectively. While COD removal performance was above 90% in all SRTs, color removal reached its lowest value at SRT of 10 d and did not show any correlation with SRT. In both MBRs, transmembrane pressure (TMP) was below 10 mbar throughout the study and no membrane fouling was observed. Supernatant filterability (SF) and specific filtration resistance (SRF) values increased at 10 d of SRT. A decrease in viscosity values was also observed due to the decrease in suspended solids (SS) concentration as SRT decreased. While SMP concentrations were similar at all tested SRT values, an increase in EPS concentration was observed at 10 d SRT. Additionally, reducing SRT resulted in an increase in waste sludge generation and observed biomass yield (Yobs), and a decrease in the energy requirement for aeration. According to gel permeation chromatography (GPC) results, as SRT decreased, organic compounds with low molecular weight had higher signals. In the second stage, aim was to investigate textile wastewater treatment performance and organic matter recovery efficiency at short SRTs, and a laboratory-scale high-rate aerobic MBR was run at SRTs of 0.5 – 5 d and HRTs of 1.2 – 24 h, corresponding to predetermined different SRT/HRT ratios of 5, 10 and 20. The average total COD, color, and soluble nitrogen concentrations in the wastewater were 834±143 mg/L and 1037±407 Pt-Co and 51±11 mg/L, respectively. While COD removal performances ranged between 86 and 92% at SRT of 5, 3, and 2 d (in all SRT/HRT ratios), it decreased to 82 and 77% at SRT 1 and 0.5 d (at SRT/HRT ratio of 10), respectively. There was no correlation between decolourization performance and SRT or HRT as it varied between 26% and 70%. The nitrification performance in the system stopped completely at SRTs ≤ 2 d. In particular, when SRT decreased from 5 days to 1 day, the amount of sludge produced and Yobs values increased. The SRT/HRT ratio played an important role in the energy requirement for aeration. In addition, reducing the SRT in the system resulted in higher SRF values, lower SF values, and rapid membrane fouling. SMP in the supernatant increased especially at SRTs ≤ 2 d. The total EPS concentration increased as SRT decreased, but the it decreased as the SRT/HRT ratio increased at each SRT value. No significant change occurred in the molecular weight distributions of the organic substances in the supernatant and filtrate at SRT of 3, 2, and 1 d. Throughout the study, in the cake layer deposited on membrane, Al, Si, and Fe were detected below 2%. In the third stage, the aim was to investigate optimum operating conditions for the simultaneous nitrification and denitrification processes to remove organic matter, color and nitrogen-based pollutants in the real textile wastewater using an MBR with an intermittent aeration strategy. The system was first operated at different dissolved oxygen values (DO of 6 and 3 mg/L) and then aeration-on/off durations varying between 2 min/2 min and 90 min/360 min. The average total COD, color, and TN concentrations of the wastewater were measured as 793±173 mg/L, 1171±458 Pt–Co and 65±15 mg/L, respectively. COD removal performance ranged from 84 to 91%. In all tested conditions, color removal performance was highly variable and independent of operating conditions, ranging from 40 to 68%. While ≥89% nitrification performance was achieved in the MBR at a minimum aeration-on durations of 30 min, the highest denitrification efficiency was achieved in the cycle with an aeration-off durations of 360 min. With the intermittent aeration process, higher TN removal, less sludge production and less energy requirement for aeration were achieved. However, membrane fouling profiles occurred more quickly at the aeration-off durations of 60 min and longer. Additionally, while SS, VSS, SF and viscosity values decreased under intermittent aeration conditions, SRF values increased. Although SMP concentrations decreased with intermittent aeration, EPS concentrations were quite similar. While no change was observed in the molecular weights of the supernatant and filtrate samples of the MBR, the average particle sizes in the supernatant increased as the aeration-off time increased. Finally, according to SEM-EDS results, inorganic substances such as Ca, Mg, Si, and Na were detected in the cake-deposited membrane surfaces. In the fourth stage, the impacts of different aeration patterns on the specific ammonium oxidation, denitritation and denitrification rates were investigated in batch assays. The batch experiments were conducted using the sludge taken from the MBR operated at various DO concentrations and aeration on/off times. While the specific ammonium oxidation rate was determined by batch tests and respirometric studies, specific denitritation and denitrification rates were determined by parallel batch reactors containing different nitrite and nitrate concentrations. The highest specific ammonium oxidation, denitritation, and denitrification rates were obtained as 5.4, 3.8, and 5.3 mg N/(g VSS.h), respectively, at the aeration-on/off durations of 90/360 min. Specific ammonium oxidation rates increased by 1.8 and 2.1 times in the last period (the aeration-on/off time of 90/360 min), compared to continuous aeration conditions with DO of 6 and 3 mg/L, respectively.
Fate, environmental impact and treatability of favipiravir and surveillance of sars-cov-2 RNA: Comparison with Covid-19 cases

(Graduate School, 2024-10-18) Yesir, Eryıldız, Bahriye ; Koyuncu, İsmail ; 501192801 ; Environmental Biotechnology

Pharmaceutical substances, such as antiviral drugs, antibiotics, antidepressants, anti-inflammatory drugs, antipyretics, beta-blockers and lipid regulators have become more common in both human and animal healthcare to enhance quality of life and prolong lifespan. This increase in pharmaceutical usage has become a significant global environmental concern in recent years. Therefore, the extensive use of these substances worldwide requires attentive monitoring to manage their contamination and environmental impact of water sources. In the second and third chapter, two reviews aimed to provide a comprehensive discussion of the physicochemical properties, analytical detection methods, removal techniques, and ecotoxicological impacts of antiviral drugs. Also, combined assessment of antiviral drugs and virus discharged in the environment were reviewed. These reviews address the challenges faced and explores future opportunities in this field of study. Special emphasis was placed on the occurrence of antiviral drugs employed in the treatment of COVID-19 in water and wastewater. In the fourth chapter, the long-term presence of favipiravir in influent, effluent wastewater, and sludge samples from two WWTPs in Istanbul were investigated. Additionally, the potential environmental risks of favipiravir were assessed using two model organisms. The study determined the mass balance, removal efficiency, and seasonal variations in favipiravir concentrations. The correlation between the number of confirmed COVID-19 cases and favipiravir concentrations in influent wastewater was also analyzed. Furthermore, the impact of drug concentration on the microbial community in sludge from various WWTPs during the pandemic was compared with post-pandemic sludge. The results demonstrate that favipiravir is partially removed (<50%) in WWTPs, with the majority of its removal mechanism being attributed to biodegradation. Additionally, a significant statistical correlation was observed between the concentration of favipiravir and the incidence of COVID-19 in Istanbul, with p-values of 0.025 and 0.039 for WWTP-1 and WWTP-2, respectively. The microbiological distribution was found to vary significantly between sludge samples taken during the COVID-19 pandemic and those from the post-pandemic period. The fifth chapter presents that the presence and quantification of SARS-CoV-2 RNA in raw wastewater, treated effluents, and secondary sludge samples were monitored between June 2021 and January 2022 at two different WWTPs in Istanbul, Turkey. The obtained data were compared with the number of COVID-19 cases and deaths in Istanbul. Additionally, the seasonal variations of SARS-CoV-2 and its relationship with TN, TP, and COD parameters were analyzed using PCA. The results indicated that secondary treatment effectively reduces SARS-CoV-2 levels, thereby mitigating the associated risks from wastewater. This study highlights a moderate correlation between the concentration of SARS-CoV-2 genes and the number of reported COVID-19 cases and deaths in Istanbul. Furthermore, a correlation was observed between the amounts of gene copies and the levels of TP and COD for both the N1 and N2 genes at the two wastewater treatment plants. In the sixth chapter, degradation kinetics of favipiravir under UV, UV/H₂O₂, and UV/Co-doped ZnS processes was investigated. The influence of initial favipiravir concentration, pH, and water matrices (distilled water, tap water, and WWTP effluent) on the degradation kinetics of favipiravir in these processes were assessed. The ecotoxicity risks of favipiravir using algae with treated solutions containing various initial FAV concentrations were evaluated. The degradation of favipiravir after 45 min was observed to be 77.3%, 100%, 89.8%, and 100% for the UV, UV/H₂O₂, UV/Co-doped ZnS, and UV/H₂O₂/Co-doped ZnS processes, respectively. In the seventh chapter was to examine how initial concentrations of antiviral drugs and sludge retention time (SRT) affect favipiravir removal efficiency in membrane bioreactor (MBR) systems. The biotransformation kinetics of favipiravir were also investigated to understand the relationship between biokinetic coefficients, initial drug concentration and SRT. Additionally, an environmental risk assessment was carried out to assess the potential risks associated with favipiravir. Favipiravir was eliminated >99% regardless of its initial concentration in MBR systems. The removal efficiency of favipiravir improved from 48.9% to 86.4% as the sludge retention time (SRT) increased from 15 days to 45 days.
Carbondioxide capturing from industrial flue gas via calcium carbonate inducing microorganisms

(Graduate School, 2025-03-19) Kolukısaoğlu, Mert ; Altınbaş, Mahmut ; 501182801 ; Environmental Biotechnology

The increase in greenhouse gases, primarily caused by human activities in the past century, has led to the effects of global climate change becoming increasingly evident. Legal regulations in Europe and neighboring countries are becoming more stringent, and new taxation systems such as the Carbon Border Adjustment Mechanism are pushing industries with high carbon emissions to seek different solutions. In the near future, where conventional flue gas treatment methods alone will be insufficient, carbon capture technologies have been improved in recent years by scientists and even industry's research and development departments. Solutions involving microorganisms allow for many options with individual benefits, such as atmospheric and closed-loop systems. Carbon capture using microorganisms, especially algae, offers a promising solution. Within the scope of this study, experiments were conducted on two algae species with coccus and filamentous morphological structures obtained from Lake Salda together with Chlorella vulgaris, Spirulina, Chlamydomonas reinhardtii species. Many analyses were conducted on these algae including growth rates, growth periods, pigment contents such as chlorophyll and carotenoid, carbonic anhydrase enzyme activity values, biochemical content, and fatty acid types. The fact that Lake Salda is in the high pH category with a pH value greater than 9 was an effective factor in including the samples obtained from here in the study. In all analyses, the pH at which the relevant algae species showed the most growth between pH 8 and 11 at 0.5 intervals was examined. The two species obtained from Lake Salda, which were subjected to the same growth conditions as other algae species using BG11 medium, were not included in further studies as they grew relatively less. In addition, the measurement of carbonic anhydrase enzyme activity was considered as a parameter at least as important as growth during species selection. The presence of the relevant enzyme that enables the gaseous carbon dioxide to be converted into dissolved gas was evaluated as an factor that needs to be developed for carbon dioxide capture studies. The species selection was made as Spirulina with the help of the Analytical Hierarchy Process method, which takes into account all other effective factors as well as growth and enzyme activity. In the second phase of the experiment, the optimum mixture ratio of algae and bacteria coculture was examined in order to increase enzyme activity. Here, the Bacillus pasteurii species that secretes the relevant enzyme was selected. In addition to 100 ml of algae and bacteria monocultures in individual sterile bottles, 3:1, 1:1, 1:3 ratios were also added to the experimental set as two sets. After 12 days of incubation, the highest enzyme activity value was measured as 1.33 mU/mg for the algae-bacteria mixture ratio of 3:1. In growth values, the same culture stood out as 2.4 g/L. The same set of experiments was run for a third set, this time with only the Zarrouk medium in a different conical flask. Here, just before the 15-day incubation period was started, CaCl2 was added to the medium so that there would be 60 mM calcium ions in the medium. Advanced experiments such as XRD analysis, SEM imaging and qPCR analysis were performed on the precipitate obtained at the end of 15 days. The coculture 3:1 mixing ratio stood out again, especially due to the high impurity in the calcite it formed. In the third phase of the experiment, while moving on to pilot scale studies, the coculture mixture ratio was decided and it was progressed in a way. First, the study was carried out in atmospheric and agitated tanks positioned side by side in 200 L volume. Incubation lasting 35 days was carried out in October and November. 22 days of this was the time spent only for the growth of microorganisms in the aquatic environment, and CaCl2 was added to the medium in the last 13 days and the calcification process was followed. At the end of the total study, the VSS concentration in the coculture increased by 1.7 times, while the VSS concentration in the monoculture increased by only 1.4 times. At the end of the study, the VSS concentration in the monoculture was measured as 1.1 g/L, and the temperature has a great effect on the relatively low result. With bubble type photobioreactor, 4 days of calcification were performed in monoculture after 4 days of growth, and 4 days of calcification were performed in coculture after 8 days of growth. In the results, 2.12 g/L VSS was observed in coculture, while 4 g/L VSS was observed in monoculture. Despite the remarkable growth in monoculture, almost equal amounts of TSS were measured at the end of both studies as 14.64 g/L in monoculture and 14.42 g/L in coculture, respectively. As a result, it was determined that much more calcite was produced with much lower biomass in coculture. This study elucidates the multifaceted potential of microalgae, specifically Spirulina sp., in diverse biotechnological applications. The comprehensive analyses and experiments conducted have provided critical insights into optimizing conditions for enhanced biomass productivity and efficient CO2 utilization. The investigation into the properties of calcium carbonate (CaCO3) and the enzyme activity of carbonic anhydrase (CA) revealed valuable information for optimizing biomass productivity and CO2 utilization. Experiments conducted in closed photobioreactors demonstrated the advantages of regulating environmental parameters to maximize algal growth and productivity. The evaluation of suspended solids and volatile suspended solids yielded critical data on the overall efficiency and sustainability of the cultivation processes. Furthermore, the structural and compositional attributes of CaCO3 were found to be essential for its application in various industrial processes. The enzyme activity studies highlighted the pivotal role of CA in facilitating CO2 capture and conversion, underscoring its potential in mitigating greenhouse gas emissions. Additionally, the extracellular polymeric substances (EPS) analysis illuminated the complex nature of the extracellular matrix, suggesting avenues for further research into biofilm formation and stability. The study also compared carbon capture via algae with conventional carbon capture and storage (CCS) technologies, noting both advantages and challenges. Despite the benefits, scaling up algae-based capture in energy-intensive sectors remains challenging. Absorption and adsorption are considered more economical and advanced methods for substantial CO2 capture. Using the Analytic Hierarchy Process (AHP) method, the study evaluated various CCS technologies based on factors such as CO2 capture capacity, cost, operational difficulties, scalability, and space requirements. Algae-based CCS technologies face significant economic and spatial challenges, with costs ranging from $702 to $1,585 per ton of CO2 captured, compared to $15 to $340 for conventional methods. In conclusion, while microalgae demonstrate significant promise in addressing environmental and energy challenges, further research and development are necessary for industrial-scale applications. Continued collaboration and research will advance the field of microalgal biotechnology, fostering sustainable development.

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